PROJECT SUMMARY/ABSTRACT Actomyosin stress fibers (SFs) enable cells to tense the extracellular matrix (ECM), a process key to cell shape determination, polarity, motility, and tissue morphogenesis. SFs within motile cells have been broadly classified into three specialized ?subtypes? (dorsal fibers, transverse arcs, and ventral fibers) that differ in their antero-posterior location and network connectivity. In addition to driving normal tissue development and homeostasis, SFs and analogous contractile structures contribute to the invasion of tumors within tissue, a notable example of which is the perivascular infiltration of the deadly brain tumor glioblastoma multiforme (GBM). It has been hypothesized that dorsal fibers, transverse arcs, and ventral fibers tense each other and the ECM in very specific ways to govern cell shape, polarity, and motility. However, this paradigm suffers from several critical limitations. For example, it has not been directly demonstrated that each SF subtype generates tension as commonly assumed, which in turn derives from a lack of direct measurement of SF mechanical properties in living cells. Additionally, while these subtypes are broadly understood to vary in the molecular motors they contain (i.e. myosin II isoforms), we know virtually nothing about how these molecular-scale differences create the contractility differences across SF subtypes. Finally, and perhaps most importantly, it is unclear whether this subtype classification is relevant to the persistent migration of cells within tissue, particularly in disease states driven by aberrant cell migration. In this proposal we address all three of these critical open questions by combining single-cell biophotonic technologies, traditional cell and molecular biology approaches, engineered culture systems, and ex vivo tissue models. A key enabling tool for these studies (which our team has pioneered over the past decade) is femtosecond laser nanosurgery (FLN), which enables us to selectively cut single SFs in living cells, thereby allowing us to deduce both the mechanical loads borne by that SF and its structural contributions to the rest of the cell. In Aim 1, we will apply FLN to selectively incise SFs from each canonical subtype to map these mechanical properties and structural contributions. We will also combine FLN with single-cell micropatterning and fluorescence-based readouts of molecular tension to determine how single SFs distribute tension throughout the cell and contribute to EGF-dependent polarization and motility. In Aim 2, we will investigate how the stoichiometry and mechanochemical properties of specific myosin II isoforms collaborate to determine the mechanical properties of the entire SF. In Aim 3, we will combine these approaches with a microfluidic model we developed with a brain-slice paradigm to determine how specific SF subtypes and the myosin isoforms therein contribute to perivascular invasion in GBM. To our knowledge, Aim 3 studies will represent the first measurements of SF mechanics and function in mammalian tissue. In summary, this project will marry innovative single-cell and culture technologies to address major open questions surrounding the microscale, biophysical mechanisms of cell shape shape, polarity, and motility.